Erratum in

Abstract

Seborrheic keratoses (SKs) are common, benign epithelial tumors of the skin that do not, or very rarely, progress into malignancy, for reasons that are not understood. We investigated this by gene expression profiling of human SKs and cutaneous squamous cell carcinomas (SCCs) and found that several genes previously connected with keratinocyte tumor development were similarly modulated in SKs and SCCs, whereas the expression of others differed by only a few fold. In contrast, the tyrosine kinase receptor FGF receptor-3 (FGFR3) and the transcription factor forkhead box N1 (FOXN1) were highly expressed in SKs, and close to undetectable in SCCs. We also showed that increased FGFR3 activity was sufficient to induce FOXN1 expression, counteract the inhibitory effect of EGFR signaling on FOXN1 expression and differentiation, and induce differentiation in a FOXN1-dependent manner. Knockdown of FOXN1 expression in primary human keratinocytes cooperated with oncogenic RAS in the induction of SCC-like tumors, whereas increased FOXN1 expression triggered the SCC cells to shift to a benign SK-like tumor phenotype, which included increased FGFR3 expression. Thus,we have uncovered a positive regulatory loop between FGFR3 and FOXN1 that underlies a benign versus malignant skin tumor phenotype.

RNAs extracted from 7 separate SK lesions, 4 separate surgically excised SCC lesions, and 3 normal, age-matched control epidermis were subjected to real-time RT-PCR for the indicated genes, with β-actin for internal normalization. SK and SCC values are expressed as folds of expression relative to the average of the 3 normal controls. Error bars denote SEM.

(A) RNAs extracted from 7 separate SK lesions, 7 separate surgically excised SCC lesions, and 3 normal, age-matched control epidermis specimens were subjected to real-time RT-PCR with oligonucleotide primers for gene expression levels of FOXN1. The β-actin mRNA level was used for internal normalization. Similar results were obtained after normalization to 36B4 in this and other experiments. Gene expression values in SK and SCC specimens are expressed as fold expression relative to the average of the 3 normal controls. We similarly analyzed 4 other SCC samples obtained by laser capture microdissection (LCM) along with surrounding control epidermis. (B) Samples as in A were analyzed for FGFR3 mRNA expression by real-time RT-PCR, with corresponding specific primers and β-actin normalization. Error bars denote SEM.

Different expression levels of FOXN1 and FGFR3 in SK versus SCC tissue samples.

Frozen sections from 3 different surgically obtained SK and SCC tissue samples were stained with H&E or analyzed for expression of FGFR3 and FOXN1 by immunofluorescence analysis with corresponding primary antibodies and using FITC- (green) and rhodamine-conjugated (red) secondary antibodies, respectively, for detection. Concomitant staining patterns of FOXN1 and FGFR3 or of FGFR3 and DAPI (used for overall nuclear counterstaining) were visualized by image overlay. Fluorescent images correspond to the areas of the H&E sections indicated by arrows. No positive fluorescence signal was obtained when primary antibodies were preincubated with corresponding blocking peptides for 2 hours prior to incubation on sections (not shown). Scale bars: 200 μm (H&E); 20 μm (fluorescence).

(A and B) Human keratinocytes were transfected with siRNAs for EGFR, ERK1 (A), FGFR3 (B), or control siRNAs, and levels of FOXN1 mRNA were measured by real-time RT-PCR. For internal normalization, here and in all subsequent analyses, 36B4 was used. (C and D) Human keratinocytes were treated with 1 ng/ml EGF or 5 ng/ml FGF9 for 24 hours, followed by real-time RT-PCR analysis (C) or immunoblotting for FOXN1 expression (D). (E) Human keratinocytes (left) and fresh human skin explants (right) were treated with DMSO or with FGFR (SU5402) or EGFR (AG478) inhibitors and analyzed by real-time RT-PCR for FOXN1. (F) Human keratinocytes were transfected with siRNAs for the indicated genes or with control siRNA. Levels of FOXN1 mRNA were assessed by real-time RT-PCR. (G) Human keratinocytes were processed for ChIP with an antibody against c-Jun and control rabbit IgG. Real-time PCR was used to amplify regions of the human FOXN1 promoter using specific primers (Supplemental Table 3). (H) Human keratinocytes were cotransfected with a FOXN1 reporter together with a c-Jun plasmid (c-Jun–CMV; ref. ) or empty vector control (CMV). Luciferase activity was determined using Renilla for normalization. (I) Human keratinocytes were cotransfected with a FOXN1 reporter with or without expression vectors for wild-type FGFR3, a constitutively active (CA) mutant (), or control. Promoter activity was measured as described above. (J) Human keratinocytes were treated as in C and D, followed by immunoblot for phosphorylated (p) and total (t) c-Jun and ERK1/2. All error bars denote SEM.

(A and B) Human keratinocytes were treated with 1 ng/ml EGF or 5 ng/ml FGF9 separately or in combination for 24 hours followed by real-time RT-PCR analysis of keratin1 (A) and involucrin (B). (C) Human keratinocytes treated as in A and B were analyzed for involucrin expression by immunoblotting with γ-tubulin as equal loading control. (D) Human keratinocytes were transfected with anti-FOXN1 siRNAs or scrambled siRNA control followed 48 hours later by real-time RT-PCR analysis to verify efficacy of gene knockdown. (E) Keratinocytes transfected with anti-FOXN1 siRNAs or scrambled siRNA control as in D were left untreated or treated with 5 ng/ml FGF9 for the last 24 hours of the experiment. Expression levels of keratin1 and involucrin were determined by real-time RT-PCR. (F) Primary human keratinocytes transfected with FOXN1 siRNA were left untreated or treated with FGF9 as in E, followed by immunoblot analysis of involucrin expression with γ-tubulin as equal loading control. Results were quantified by densitometric scanning of the immunoblots and normalization for γ-tubulin. All error bars denote SEM.

(A) The keratinocyte-derived SCC cell lines SCC13 and SCCO28 were infected with a retrovirus expressing FOXN1-ER fusion protein () or empty vector control. Cells with or without 200 nM 4-OHT treatment for 24 hours were analyzed, in parallel with control human keratinocytes, for levels of FOXN1 expression by immunoblotting, with γ-tubulin for equal loading control. Endogenous FOXN1 was detected at 69 kDa, and the higher–molecular weight band in the SCC cells corresponds to expression of the FOXN1-ER fusion protein. Lanes were run on the same gel but were noncontiguous (white line). (B–D) SCC13 and SCCO28 cells infected with the FOXN1-ER retrovirus or empty vector control were treated with 200 nM 4-OHT for 24 hours to induce activation of FOXN1-ER. Expression of involucrin (B), keratin1 (C), and FGFR3 (D) was analyzed by real-time RT-PCR using 36B4 for normalization. Error bars denote SEM. (E) SCCO28 cells were transduced with a FOXN1-expressing adenovirus () or GFP control and analyzed for expression of FGFR3 and keratin1 by immunoblotting, with γ-tubulin for equal loading control. Results were quantified by densitometric scanning of the immunoblots and normalization for γ-tubulin.

(A) Human keratinocytes infected with 2 shRNA constructs targeting FOXN1 expression (shFOXN1) in parallel with empty vector control, were superinfected with a H-rasV12–expressing retrovirus (). Cells (1 × 106) were injected at the dermal-epidermal junction of the back skin of nude mice. Tissue samples from 4 individual tumors and 2 controls were analyzed by immunohistochemistry with anti–pan-keratin and anti–human-specific vimentin antibodies. (B and C) SCC13 cells infected with a FOXN1-ER–expressing retrovirus or empty vector control were injected in parallel into the right and left suprascapular areas of NOD/SCID mice (5 × 105 cells per injection), After 1 week, animals were treated with 200 μg 4-OHT (see Methods) or vehicle control by i.p. injections for 5 weeks. Tumors (7 per condition) were separated from surrounding tissue for weight determination (B), and tissue samples were processed for H&E analysis (C). Similar results were obtained in a second similar experiment. Error bars denote SEM. Scale bars: 100 μm.

Increased FOXN1 expression converts the gene signature of tumors formed by SCC cells.

The 7 individual pairs of tumors, obtained as described in Figure by parallel injections of mice with FOXN1-ER–expressing SCC13 cells versus controls, were analyzed by real-time RT-PCR for expression of the indicated genes. In each case, gene expression levels in the tumor formed by FOXN1-ER–expressing cells are shown relative to those in the tumor formed by control cells in the same mouse. Error bars denote SEM.